Human coagulation factor X deficiency caused by a mutant signal peptide that blocks cleavage by signal peptidase but not targeting and translocation to the endoplasmic reticulum

Human factor XSanto Domingo is a form of coagulation factor X in which a mutation within the signal peptide region of the precursor protein has been correlated genetically with a severe deficiency of factor X in the affected individual. A point mutation results in substitution of Arg for Gly at the critical -3 position of the factor X signal peptide. To determine the biochemical effect of this mutation on the biosynthesis of factor X, the wild-type and mutant factor X cDNAs were subcloned into a vector for transcription and translation in vitro. Translation products of mRNAs encoding portions of both mutant and wild-type proteins were used in a systematic biochemical approach to evaluate directly the effect of the mutation on targeting, transport, and proteolytic processing in vitro. The results show that targeting and transport of factor XSanto Domingo to the endoplasmic reticulum are functionally dissociated from the removal of the signal peptide by signal peptidase. Factor XSanto Domingo is translocated into the endoplasmic reticulum but not processed by signal peptidase. Transient expression of the wild-type and mutant factor X in human embryonic kidney 293 cells revealed apparently normal secretion of the glycosylated two-chain form of factor X but no secretion of factor XSanto Domingo. Thus, the inability of signal peptidase to cleave factor XSanto Domingo is directly responsible for the absence of circulating factor X and leads to the bleeding diathesis in the affected individual

Multidetector CT imaging of mechanical prosthetic heart valves: quantification of artifacts with a pulsatile in-vitro model

Item does not contain fulltextOBJECTIVES: Multidetector computed tomography (MDCT) can detect the cause of prosthetic heart valve (PHV) dysfunction but is hampered by valve-induced artifacts. We quantified artifacts of four PHV using a pulsatile in-vitro model and assessed the relation to leaflet motion and valve design. METHODS: A Medtronic Hall tilting disc (MH), and Carbomedics (CM), St Jude (SJM), and ON-X bileaflet valves underwent CT in an in-vitro model using retrospective gating with a 64 detector CT system in stationary and pulsatile conditions. Artifacts and radiopaque component volumes were quantified with thresholds based on surrounding structures and valvular components. RESULTS: Hypodense artifacts volumes (mm(3)) were 1,029 +/- 147, 535 +/- 53, 371 +/- 16, and 366 +/- 18 for the SJM, MH, CM and ON-X valves (p < 0.001 except for the latter two valves p = 0.43). Hyperdense artifact volumes were 3,546 +/- 141, 2,387 +/- 103, 2,003 +/- 102, and 3,033 +/- 31 for the SJM, MH, CM and ON-X valve, respectively (all differences p < 0.001). Leaflet motion affected hypodense (F = 41.5, p < 0.001) and hyperdense artifacts (F = 53.7, p < 0.001). Closed and moving leaflets were associated with the least and the most artifacts respectively (p < 0.001, both artifact types). CONCLUSION: Both valve design and leaflet motion affect PHV-induced artifacts. Best imaging results may be expected for the CM valve during phases in which the leaflets are closed

Measuring femoral lesions despite CT metal artefacts: a cadaveric study

Objective Computed tomography is the modality of choice for measuring osteolysis but suffers from metal-induced artefacts obscuring periprosthetic tissues. Previous papers on metal artefact reduction (MAR) show qualitative improvements, but their algorithms have not found acceptance for clinical applications. We investigated to what extent metal artefacts interfere with the segmentation of lesions adjacent to a metal femoral implant and whether metal artefact reduction improves the manual segmentation of such lesions. Materials and methods We manually created 27 periprosthetic lesions in 10 human cadaver femora. We filled the lesions with a fibrotic interface tissue substitute. Each femur was fitted with a polished tapered cobalt-chrome prosthesis and imaged twice—once with the metal, and once with a substitute resin prosthesis inserted. Metalaffected CTs were processed using standard back-projection as well as projection interpolation (PI) MAR. Two experienced users segmented all lesions and compared segmentation accuracy. Results We achieved accurate delineation of periprosthetic lesions in the metal-free images. The presence of a metal implant led us to underestimate lesion volume and introduced geometrical errors in segmentation boundaries.MediamaticsElectrical Engineering, Mathematics and Computer Scienc

Backpack-mounted satellite transmitters do not affect reproductive performance in a migratory bustard

Backpack-mounted satellite transmitters (PTTs) are used extensively in the study of avian habitat use and of the movements and demography of medium- to large-bodied species, but can affect individuals’ performance and fitness. Transparent assessment of potential transmitter effects is important for both ethical accountability and confidence in, or adjustment to, life history parameter estimates. We assessed the influence of transmitters on seven reproductive parameters in Asian houbara Chlamydotis macqueenii, comparing 114 nests of 38 females carrying PTTs to 184 nests of untagged birds (non-PTT) over seven breeding seasons (2012‒2018) in Uzbekistan. There was no evidence of any influence of PTTs on: lay date (non-PTT x̅ = 91.7 Julian day ± 12.3 SD; PTT x̅ = 95.1 Julian day ± 15.7 SD); clutch size (non-PTT x̅ = 3.30 ± 0.68 SD; PTT x̅ = 3.25 ± 0.65 SD); mean egg weight at laying (non-PTT x̅ = 66.1g ± 5.4 SD; PTT x̅ = 66.4g ± 5.4 SD); nest success (non-PTT x̅ = 57.08% ± 4.3 SE; PTT x̅ = 58.24% ± 4.5 SE for nests started 2 April); egg hatchability (non-PTT x̅ = 88.3% ± 2.2 SE; PTT x̅ = 88.3% ± 2.6 SE); or chick survival to fledging from broods that had at least one surviving chick (non-PTT x̅ = 63.4% ± 4.2 SE; PTT x̅= 64.4% ± 4.7 SE). High nesting propensity (97.3% year-1 ± 1.9% SE) of tagged birds indicated minimal PTT effect on breeding probability. These findings show harness-mounted transmitters can give unbiased measures of demographic parameters of this species, and are relevant to other large-bodied, cursorial, ground-nesting birds of open habitats, particularly other bustards

A randomised controlled trial investigating the effect of nutritional supplementation on visual function in normal, and age-related macular disease affected eyes: design and methodology [ISRCTN78467674]

BACKGROUND: Age-related macular disease is the leading cause of blind registration in the developed world. One aetiological hypothesis involves oxidation, and the intrinsic vulnerability of the retina to damage via this process. This has prompted interest in the role of antioxidants, particularly the carotenoids lutein and zeaxanthin, in the prevention and treatment of this eye disease. METHODS: The aim of this randomised controlled trial is to determine the effect of a nutritional supplement containing lutein, vitamins A, C and E, zinc, and copper on measures of visual function in people with and without age-related macular disease. Outcome measures are distance and near visual acuity, contrast sensitivity, colour vision, macular visual field, glare recovery, and fundus photography. Randomisation is achieved via a random number generator, and masking achieved by third party coding of the active and placebo containers. Data collection will take place at nine and 18 months, and statistical analysis will employ Student's t test. DISCUSSION: A paucity of treatment modalities for age-related macular disease has prompted research into the development of prevention strategies. A positive effect on normals may be indicative of a role of nutritional supplementation in preventing or delaying onset of the condition. An observed benefit in the age-related macular disease group may indicate a potential role of supplementation in prevention of progression, or even a degree reversal of the visual effects caused by this condition

Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher’s disease. More recently, mutations in the&nbsp;GBA1&nbsp;gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson’s disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging

Competition between lipases and monoglycerides at interfaces

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